Neurotoxic oligomers

ABSTRACT

This invention relates to methods and compositions for the treatment or alleviation of Alzheimer&#39;s disease and of other conditions related to abnormal protein aggregation. In particular, the invention relates to methods and compositions for the immunotherapy of Alzheimer&#39;s disease, Parkinson&#39;s disease, and cataract. In one aspect the invention provides a method of prophylaxis, treatment or alleviation of a condition characterized by pathological aggregation and accumulation of a specific protein associated with an immunizing-effective dose of one or more tyrosine cross-linked compounds, and optionally also comprising copper ions complexed to the compound. Alternatively passive immunization against a tyrosine cross-linked compound may be used. Prophylactic or therapeutic compositions and diagnostic methods are also disclosed and claimed.

FIELD OF THE INVENTION

[0001] This invention relates to methods and compositions for thetreatment or alleviation of Alzheimer's disease and of other conditionsrelated to abnormal protein aggregation. In particular, the inventionrelates to methods and compositions for the immunotherapy of Alzheimer'sdisease, Parkinson's disease, and cataract.

BACKGROUND OF THE INVENTION

[0002] The characteristic amyloid lesions of Alzheimer's disease (AD)are primarily composed of Amyloid β (Aβ) (Glenner & Wong, 1984), a 39-43amino acid protein which is a normally soluble protein found inbiological fluids. disease, so identifying the neurochemical changeswhich lead to the inhibition of Aβ catabolism and its accumulation inthe neocortex would be an important clue to the pathogenesis of AD.

[0003] Although the fundamental pathology, genetic susceptibility andbiology associated with AD are becoming clearer, a rational chemical andstructural basis for developing effective drugs to prevent or cure thedisease remains elusive. While the genetics of AD indicate that themetabolism of Aβ is intimately associated with the pathogenesis of thedisease as indicated above, drugs for the treatment of AD have so farfocused on “cognition enhancers”, which do not address the underlyingdisease. processes. These drugs have met with only limited success.

[0004] The nature of the deranged neurochemical environment in AD can bepartly deduced from the post-translational modifications of amyloid Aβ.Aβ extracted from biological systems normally migrates as an apparent ˜4kD monomer on sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE; (Shoji et al., 1992)); however, Aβ extracted from specimensof AD-affected post-mortem brain migrates on SDS-PAGE as SDS-, urea- andformic acid-resistant oligomers (Masters et al., 1985; Roher et al.,1996; Cherny et al., 1999).

[0005] Matrix-assisted laser desorption ionization-mass spectrometry(MALDI-MS) of these SDS-resistant oligomers extracted from neuriticplaque and vascular amyloid indicates the presence of covalentlycross-linked dimeric and trimeric Aβ species (Roher et al., 1996).

[0006] Synthetic Aβ₁₋₄₀ and Aβ₁₋₄₂ normally migrate as apparent monomerson SDS-PAGE, but form apparent higher molecular weight species uponincubation (Burdick et al., 1992). This process is accelerated byexposure to oxidative systems (Dyrks et al., 1992; Atwood et al., 1997).

[0007] Tyrosine cross-linking has been proposed as a mechanism of Aβoligomerization in vivo, since tyrosine residues in synthetic human Aβcan be cross-linked by peroxidase-catalyzed oxidation systems (Galeazziet al., 1999). As Rat Aβ, unlike human Aβ, lacks a tyrosine residue(Atwood et al.,1997), it is therefore resistant to metal-catalyzedoxidative oligomerization, and this perhaps explains the rarity ofamyloid deposits in these animals (Vaughan and Peters, 1981).

[0008] Tyrosine cross-linking in proteins is a sensitive marker ofoxidative stress. Covalent carbon-carbon bridges or carbon-oxygenbridges are formed between single tyrosyl residues and/or dityrosylresidues, resulting in a number of stable, fluorescent reaction products(Gross and Sizer, 1959; Amado et al., 1984, Jacob et al., 1996). Themajor reaction products of the free tyrosyl radical are the intenselyfluorescent amino acids 3,3′-dityrosine (DT), 3,3′,3′-trityrosine (TT)and pulcherosine (P), and the non-fluorescent isodityrosine (iso-DT)(Gross and Sizer, 1959; Amado et al., 1984, Jacob et al., 1996; Heineckeet al., 1993). DT and 3-nitrotyrosine levels are elevated in thehippocampus and neocortical regions of brains of patients with ADcompared to the same regions of normal brain, and are also elevated inventricular cerebrospinal fluid in AD patients (Hensley et al., 1998).

[0009] Tyrosine cross-linking may also be important in otherneurodegenerative diseases such as Parkinson's disease, and otherconditions in which α-synuclein fibrils are deposited. These includeParkinson's disease itself, dementia with Lewy body formation, multiplesystem atrophy, Hallerboden-Spatz disease, and diffuse Lewy bodydisease. Exposure of recombinant α-synuclein to nitrating agents resultsin nitration of tyrosine residues as well as oxidation of tyrosine toform DT; this results in cross-linking of α-synuclein to form stableaggregates (Souza et al, 2000). The same authors also found thatmonoclonal antibodies raised against nitrated synuclein boundspecifically to Lewy bodies and to glial cell inclusions in a variety ofsynucleinopathies (Duda et al., in preparation referred to in Souza etal., 2000).

[0010] We have now found that human amyloid-derived Aβ contains tyrosinecross-links, and includes both dityrosine and trityrosine cross-linkedspecies. These cross-links can be replicated in vitro, for example byincubating synthetic human Aβ with peroxidase and H₂O₂, or with H₂O₂ inthe presence of copper ions. These modifications are protease-resistant,and therefore we propose that tyrosine cross-linkage in AD caused byabnormal interaction of Aβ with H₂O₂ and peroxidases or copper ionscontributes to the formation of neurotoxic Aβ oligomers, and to thedeposition of Aβ. Immunization against low molecular weight tyrosinecross-linked compounds rather than with whole Aβ can therefore be usedfor treatment or prevention of AD, without the risk of provokingautoimmune complications which could otherwise be induced byimmunization with intact Aβ or large fragments thereof. By restrictingthe target for immunotherapy to an abnormal fragment or portion of themolecule, it may be possible to minimise undesirable interference withthe normal function of the molecule, while providing an active therapyagainst the abnormal molecule. It will be appreciated that either activeor passive immunization may be used.

[0011] The oxidative processes which give rise to covalent cross-linkingof proteins via tyrosine are also associated with other disorders whichare characterised by pathological aggregation and accumulation ofspecific proteins. It is therefore considered that these conditions alsowill be amenable to prevention or treatment by the method of theinvention.

[0012] It will be clearly understood that, although a number of priorart publications are referred to herein, this reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in Australia or in any other country.

SUMMARY OF THE INVENTION

[0013] In a first aspect, the invention provides a method ofprophylaxis, treatment or alleviation of a condition, in which thecondition is characterised by pathological aggregation and accumulationof a specific protein associated with oxidative damage and formation oftyrosine cross-links, the method comprising the step of immunizing asubject in need thereof with an immunizing-effective dose of one or morecompounds selected from the group consisting of dityrosine, trityrosine,tetratyrosine (also known as pulcherosine), oxidised tyrosineorthologues such as o-tyrosine and m-tyrosine, nitrotyrosine, andpeptides comprising tyrosine cross-links, and optionally also comprisingcopper ions complexed to the compound. These compounds are collectivelyreferred to herein as “tyrosine cross-linked compounds”.

[0014] A person of ordinary skill in the art will recognise that animmunizing-effective dose of the compound is one which will elicitantibody which is able to bind to a tyrosine cross-linked compound. Sucha person will also be able to determine whether a particular tyrosinecross-linked compound elicits an antibody.

[0015] In a preferred embodiment, the pathologically aggregated form ofthe specific protein comprises a tyrosine cross-linked moiety. In aparticularly preferred embodiment, the tyrosine cross-linked compound isa peptide which is an immunogenic portion of the pathologicallyaggregated form of the specific protein, the peptide comprising across-linked tyrosine moiety linked to residues upstream and downstreamof the cross-linked tyrosine.

[0016] In a preferred embodiment, the tyrosine cross-linked compound isa dityrosine cross-linked compound.

[0017] Up to 3 equivalents of copper per equivalent of dityrosine may beused, provided that each dose administered contains no more than 1 μMcopper.

[0018] Optionally the compound used for immunisation is coupled to acarrier protein which is itself immunogenic, such as tetanus toxoid,keyhole limpet haemocyanin, or albumin. Also optionally the compound maybe administered together with an adjuvant such as alum, monophosphoryllipid, a muramyl peptide, an iscom such as QS21 and the like. Personsskilled in the art will be well aware of suitable carriers andadjuvants.

[0019] Where a peptide comprising tyrosine cross-links is used, this ispreferably a minimal and immunogenic portion of the particular proteinassociated with the condition, which is constituted by the dityrosinemoiety linked to residues upstream and downstream of the cross-linkedtyrosine. Where the condition is Alzheimer's disease, preferably thepeptide comprising tyrosine cross-links is derived from the sequencesurrounding tyrosine 10 in the amino acid sequence of human Aβ₁₋₄₀ orAβ₁₋₄₂.

[0020] In all aspects of the invention, where a peptide comprisingtyrosine cross-links is used, it is preferred that the tyrosinecross-links are obtainable by oxidation in the presence of copper ions.

[0021] More preferably the peptide also comprises copper ions complexedto dityrosine.

[0022] Immunization may be administered by any convenient route,including subcutaneous, intramuscular or intravenous injection,application to mucosal surfaces, or topical administration, for examplein an ointment.

[0023] The dose of the compound to be administered will vary, dependingon the nature of the individual compound, the weight, age and generalstate of health of the patient, and whether an adjuvant is used. It iscontemplated that the dose will be in the region of 0.1 μg to 200 mg ofDT, more preferably 1 to 50 mg, most preferably 10 to 20 mg. Although asingle immunization may be given, preferably multiple immunizations areadministered, for example once a week for one to twelve months, morepreferably for four months. A booster series may be given after six totwelve months. The immune response is monitored by measuring DTantibodies; any convenient assay system may be used, such as ELISA.

[0024] In an optional embodiment, the method also comprises theadditional steps of identifying the predominant forms of the tyrosinecross-links in the pathologically aggregated specific protein; andsynthesising one or more tyrosine cross-linked compounds comprising oneor more of the predominant forms of tyrosine cross-links.

[0025] In an alternative form of this aspect of the invention, theimmunization may be passive. Thus the invention provides a method of amethod of prophylaxis, treatment or alleviation of a condition, in whichthe condition is characterised by pathological aggregation andaccumulation of a specific protein associated with oxidative damage andwhere the pathologically aggregated form of the specific proteincomprises a tyrosine cross-link, the method comprising the step ofadministering an effective amount of an antibody or an antibodyfragment,

[0026] said antibody or antibody fragment is raised against a tyrosinecross-linked compound,

[0027] said compound being an immunogenic portion of the pathologicallyaggregated form of the specific protein and comprising a tyrosinecross-link ,

[0028] and which antibody or antibody fragment is capable ofspecifically binding the pathologically aggregated form of the specificprotein,

[0029] to a subject in need of such treatment.

[0030] The antibody may be polyclonal or monoclonal. Where the antibodyis polyclonal, it is preferably of human origin, and may for example bederived from pooled human serum from normal healthy individuals.Alternatively serum from individuals who have been hyperimmunizedagainst a tyrosine cross-linked compound may be used. Protocols forhyperimmunization are known in the art. The antibody may be isolatedfrom serum by any convenient method; a variety of suitable methods isknown in the art. Where the antibody is monoclonal, it is preferablyhumanized. It will be clearly understood that antigen-binding fragmentsof antibodies, such as F(ab′), F(ab′)₂, Fv or monoclonal scFv, arewithin the scope of the invention. Methods for production andpurification of polyclonal and monoclonal antibodies and for recombinantproduction of humanized monoclonal antibodies or of scFv fragments arewell known in the art. See for example Harlow and Lane (1988);WO90/07861; and WO92/01047. Humanized monoclonal antibodies may also beproduced in transgenic mammals; see for example WO91/10741 andWO93/12227.

[0031] It is preferred that the antibody reacts specifically with thepathologically aggregated form of the specific protein, and does notreact significantly with the unaggregated form of the protein.

[0032] Following either active or passive immunization, the patient ismonitored for clinical improvement, which may commence within as littleas one week, but more probably may be observed at six weeks, and maytake as long as 12 months. The normal clinical indices which are used inthe monitoring of patients with the relevant condition are used. Theattending clinician will be aware of the most suitable tests to use.

[0033] Where the treatment is prophylactic, the patient is monitored forsigns of development of the condition. The patient may be at risk as aresult of genetic linkage, e.g. in familial Alzheimer's disease orHuntington's disease.

[0034] In a second aspect, therefore, the invention provides aprophylactic or therapeutic composition for use in the method of theinvention, comprising a tyrosine cross-linked compound, together with apharmaceutically acceptable carrier, and optionally further comprisingan adjuvant, and/or copper ions complexed to the compound.

[0035] In an alternative embodiment of the second aspect, the inventionprovides a prophylactic or therapeutic composition for use in thepassive immunization method of the invention, comprising an antibodydirected against a tyrosine cross-linked compound as defined above, or afragment thereof which is capable of binding to the tyrosinecross-linked compound, together with a pharmaceutically acceptablecarrier.

[0036] In a third aspect, the invention provides a method of diagnosisof a condition, in which the condition is characterised by pathologicalaggregation and accumulation of a specific protein associated withoxidative damage and formation of tyrosine cross-links, the methodcomprising the step of assaying a sample of a biological fluid from asubject suspected of suffering from the condition for the presence of acompound selected from the group consisting of dityrosine, trityrosine,tetratyrosine, oxidised tyrosine orthologues such as o-tyrosine andm-tyrosine, nitrotyrosine, and peptides comprising tyrosine cross-links.

[0037] In an alternative aspect, the method comprises the step ofassaying a biological fluid from a subject suspected of suffering fromthe condition for the presence of antibody directed against a tyrosinecross-linked compound.

[0038] Preferably the biological fluid is selected from the groupconsisting of blood, plasma, serum, cerebrospinal fluid, urine, andsaliva. Preferably the compound is dityrosine.

[0039] The assay may be performed by any suitable means, but is mostconveniently performed by an ELISA assay using antibody directed againsttyrosine cross-linked compounds. Such an assay may conversely be used todetect antibody directed against a tyrosine cross-linked compound.Preferably the antibody is a monoclonal antibody, or a mixture ofmonoclonal antibodies. Alternatively the assay may be performed bymeasuring fluorescence at an excitation wavelength of 325 nm and anemission wavelength of 350-500 nm.

[0040] In all three aspects of this invention, preferably the conditionis selected from the group consisting of Alzheimer's disease,amyotrophic lateral sclerosis, motoneuron disease, cataract, Parkinson'sdisease, Creutzfeldt-Jacob disease, Huntington's disease, dementia withLewy body formation, multiple system atrophy, Hallerboden-Spatz disease,and diffuse Lewy body disease, or cataract.

[0041] More preferably the condition is Alzheimer's disease orParkinson's disease.

[0042] For the purposes of this specification it will be clearlyunderstood that the word “comprising” means “including but not limitedto”, and that the word “comprises” has a corresponding meaning.

BRIEF DESCRIPTION OF THE FIGURES

[0043]FIG. 1 shows that human Aβ, but not rat Aβ, develops fluorescenceand SDS-resistance following peroxidase-catalyzed oxidation. HumanAβ₁₋₄₀, human Aβ₁₋₄₂, or rat Aβ₁₋₄₀ (50 μM) was incubated in 50 mMborate, pH 9.5±H₂O₂(1 mM) and peroxidase (7.5 μg/ml), for 1 day at 37°C.

[0044] (A) fluorescent spectra (λ_(ex) 325, λ_(em) 350-500);

[0045] (B) migration on SDS-PAGE (by Western blot using 4G8);

[0046] (C) Aβ₁₋₄₂ (10 nM) was incubated with H₂O₂ (1 μM) and peroxidase(7.5 μg/ml) for 5 days at 37° C. in phosphate buffered saline, pH 7.4.The product (lane 2) was compared to peptide incubated under the sameconditions in the absence of H₂O₂ /peroxidase (lane 1) by SDS PAGE andWestern blot (4G8)

[0047]FIG. 2 shows that human amyloid-derived Aβ contains tyrosinecross-linked oligomers. Human amyloid-derived Aβ (20 μM) (Roher et al.,1996) was analysed by fluorescence spectroscopy compared to a pure DTstandard (λ_(ex) 325, λ_(em) 350-500) (A), and Western blot (4G8) (B).

[0048]FIG. 3 shows that dityrosine and trityrosine cross-links arepresent in human amyloid-derived Aβ, and that they bind copper.

[0049] (A) and (B) Human amyloid was purified, hydrolyzed and the massspectrum determined after chromatographic separation. Two individualscans reflecting analyses of the same sample eluting at differentchromatographic retention times (RT) are shown.

[0050] (C) Absorbances at 280 nm and 315 nm of purified DT in thepresence of increasing concentrations of CuSO₄ or NaCl.

[0051]FIG. 4 shows that soluble human Aβ binds copper with highaffinity.

[0052] (A) Silver stain of crude soluble extract (1) and pH 1 eluatefrom the copper-chelating Sepharose column (2).

[0053] (B) Western blot of pH 1 eluate probed with WO2, G211 and G210.

[0054]FIG. 5 shows the results of LC-MS analysis, confirming that humanAβ binds copper.

[0055] LC-MS analysis of crude (5A) and IMAC purified (5B) solubleextracts.

[0056] Mass spectra of Aβ₁₋₄₂ (5C) and (5E), and Aβ₁₋₄₀ with two boundcopper atoms (5D) and (5F).

[0057] The IMAC and LC-MS data demonstrate that brain-derived Aβ canbind copper.

[0058]FIG. 6 shows the detection of dityrosine in cross-linked Aβ₁₋₄₀and Aβ₁₋₄₂ in Western blots.

[0059] Two techniques to create the dityrosine linkages are alsocompared.

[0060] The top Western blot (A) demonstrates the presence of Aβ usingthe WO2 antibody. The bottom blot (B) demonstrates the presence ofdityrosine linkages recognised by the monoclonal antibody IC3. Thisantibody was raised against a form of dityrosine prepared usingborate/H₂O₂/horseradish peroxidase. Lane 1 Aβ₁₋₄₀-borate cross linkingLane 2 Aβ₁₋₄₂-borate cross linking Lane 3 Aβ₁₋₄₀-copper cross linkingLane 4 Aβ₁₋₄₂-copper cross linking Lane 5 Aβ₁₋₄₀-untreated Lane 6Aβ₁₋₄₂-untreated Lane 7 Dityrosine conjugated to KLH

[0061]FIG. 7 shows examples of the forms of tyrosine cross-linksproduced as potential immunogens. These structures contain tyrosinecross-links and have the carboxy- and amino-termini acetylated to mimicthe presence of additional amino acid residues that would normally bepresent on either side of a tyrosine cross-linked moiety in a tyrosinecross-linked peptide. The presentation of multiple copies of thedityrosine antigen is designed to improve the strength of the immuneresponse generated. 7A Tyrosine 7B Dityrosine 7C Atee 7D DiAtee 7EIsoDiAtee 7F TriAtee 7G TetraAtee 7H Alternate form of TriAtee with oneiso bond.

[0062]FIG. 8 shows the detection of dityrosine bonds in a variety oftyrosine cross-linked species in Western Blots. The DT-containingspecies include dityrosine cross-linked Aβ₉₋₁₆ dimer or trimer linked toBSA, and various poly-DT species linked to either BSA or KLH carrierproteins. The top Western blot (A) demonstrates the ability of thesample to bind to a polyclonal rabbit antiserum raised against DT whichwas prepared using the borate/H₂O₂/peroxidase technique and linked toKLH using glutaraldehyde (discussed in Example 7). The bottom Westernblot (B) demonstrates the presence of dityrosine linkages recognised bythe monoclonal antibody IC3. This antibody was raised against a form ofdityrosine also prepared using the borate/H₂O₂/peroxidase technique.Lane 1 Abeta 9-16 DT dimer-BSA Lane 2 Abeta 9-16 DT trimer-BSA Lane 3Crude ATEE-BSA Lane 4 PolyTyr-BSA Lane 5 BSA Lane 6 Abeta trimer-KLHLane 7 Crude ATEE-KLH Lane 8 PolyTyr-KLH Lane 9 KLH

DETAILED DESCRIPTION OF THE INVENTION

[0063] The invention will now be described in detail by way of referenceonly to the following non-limiting examples and drawings.

[0064] Abbreviations used herein are as follows: AD Alzheimer's diseaseDT 3,3′-dityrosine TT 3,3′3′-trityrosine P pulcherosine iso-DTisodityrosine

Experimental Procedures

[0065] Reagents and Aβ Peptide Preparation

[0066] Oligomeric Aβ was extracted from amyloid plaques of humanAD-affected brains as previously described (Roher et al., 1996). Thepurified amyloid Aβ was solubilized in formic acid, and then immediatelydialyzed with 5 changes of 100 mM ammonium bicarbonate, pH 7.5 beforeuse.

[0067] Human Aβ₁₋₄₀, Aβ₁₋₄₂ and rat Aβ₁₋₄₀ were synthesized, purifiedand characterized by HPLC analysis, amino acid analysis and massspectroscopy by W. M. Keck Foundation Biotechnology Resource Laboratory(Yale University, New Haven, Conn.), and corroborative studies wereperformed using peptide synthesized by Quality Control Biochemicals,Inc. (Hopkinton, Mass.).

[0068] Each peptide was identified as a single peak by HPLC. SyntheticAβ peptides were dissolved in doubly deionized water at a concentrationof 0.5-1.0 mg/ml, sonicated for 3 min. and then centrifuged for 20 min.at 10 000 g and the supernatant (stock Aβ) used on the day of theexperiment. The concentrations of stock Aβ peptides were determined byspectrophotometric absorbance at 214 nm or by Micro BCA protein assay(Pierce, Rockford, Ill.) as previously described (Atwood et al., 1998).

[0069] Prior to use, all buffers and stock solutions of metal ions werefiltered though a 0.22 μm filter (Gelman Sciences, Ann Arbor, Mich.) toremove particulate matter. All other reagents were analytical grade orpurer. Horseradish peroxidase was obtained from Sigma Chemical Co. (St.Louis, Mo.).

[0070] Preparation and Fluorescence Analysis of Dityrosine and TyrosineCross-Linked Aβ

[0071] DT standards were generated by incubating L-tyrosine (1 mg/ml)solubilized in borate buffer (50 MM, pH 9.5) with H₂O₂ (5 mM) andhorseradish peroxidase (7.5 μg/ml) for 1 day at 37° C. (Amado et al.,1984).

[0072] Cross-linked Aβ was generated by incubating Aβ (50 μM) in boratebuffer (50 mM, pH 9.5) and with H₂O₂ (1 mM) and peroxidase (7.5 μg/ml)for 5 days at 37° C. In a separate experiment to study this reactionunder conditions which approached physiological, Aβ₁₋₄₂ was diluted to10 nM in phosphate-buffered saline (PBS, pH 7.4), and incubated with 1μM H₂O₂ and peroxidase (7.5 μg/ml) for 5 days at 37° C. Following theincubation, the samples were lyophilized to bring the peptide into aconcentration range which could be detected by Western blot (see below).

[0073] Reaction products were separated by fast phase liquidchromatography (FPLC). Excess borate was first precipitated from samplesprior to chromatography by centrifugation at 0° C. Samples were thenacidified by addition of 0.25% TFA and remaining insoluble materialremoved by filtration (0.22 μm pore size). Samples were loaded on to a 3ml Resource RPC column (Pharmacia, Uppsala, Sweden) and the columnwashed with water containing 0.1% TFA. Bound species were eluted with a0-100% linear gradient of acetonitrile containing 0.1% TFA at 1 ml/minover 45 min and collected in 0.5 ml fractions. Fractions were dried,reconstituted in water and assayed for dityrosine by fluorescence(excitation 330 nm; emission 400 nm) and UV absorbance (284 nm). Peakfractions were further characterized by mass spectrometry, anddityrosine quantitated using the extinction coefficient (E₃₁₅ nm=8380M⁻¹ cm⁻¹; Malencik et al., 1996).

[0074] Solutions were analyzed for the presence of fluorescent compoundsusing a Hitachi F-4500 spectrofluorometer. DT, TT and P havecharacteristic emission spectra (λ_(ex) 325 nm, λ_(em) 350-500 nm),which are quite distinct from those of tyrosine and tryptophan, which donot fluoresce at these wavelengths. There was a linear increase influorescence at this emission range with increasing dityrosineconcentration between 0-50 μM.

[0075] MALDI-TOF Mass Spectrometry

[0076] Samples of SDS-resistant, oligomeric, human amyloid-derived Aβwere hydrolyzed in vacuo with 6N HCl for 48 h at 105° C. Following this,samples were analyzed by liquid chromatography MALDI-TOP massspectrometry (LC-MS) at the Harvard University Mass SpectrometryFacility.

[0077] Mass spectra were obtained using a LCT mass spectrometer(Micromass Inc, Beverly Mass.) interfaced with a HP 1100 liquidchromatograph, attached to a C18 reversed-phase column (2.1 mm×250 mm).LC-MS was performed using a gradient of buffer A (water-0.1% formic acid(FA)), and buffer B (acetonitrile-0.1% FA). The gradient was from 2% B(0-2 min), to 100% B (20-23 min).

[0078] Western Blot Analysis

[0079] Aliquots of each reaction (2 ng peptide) were collected into 15μl sample buffer (containing 4% SDS, 5% β-mercaptoethanol) and heated to95° C. (5 min). Samples were run on PAGE (Tricine gels, 10-20%; Novex,San Diego, Calif.), transferred to PVDF membranes (Bio-Rad Laboratories,Hercules, Calif.), fixed with glutaraldehdye (1%, v/v), blocked withmilk (10%, w/v) and then probed with the anti-Aβ monoclonal antibody 4G8(Senetek, Maryland Heights, Mich.) overnight at 4° C. In one experimentthe monoclonal antibodies WO2 (epitope:residues 5-8), G211(epitope:residues 35-42) or G210 (epitope:residues 33-40) were used. Theblot was then incubated with anti-mouse horseradish peroxidase (HRP)conjugate (Pierce, Rockford, Ill.) for 2 h at room temperature, anddeveloped with ECL reagent (Amersham, Little Chalfont, UK) orSupersignal Ultra (Pierce, Rockford, Ill.). The chemiluminescent signalwas captured using the Fluoro-S Image Analysis System (Bio-Rad,Hercules, Calif.) and electronic images analyzed using Multi-AnalystSoftware (Bio-Rad, Hercules, Calif.). Molecular size markers were fromAmersham (Arlington Heights, Ill.).

EXAMPLE 1 Peroxidase-Catalyzed Aβ Polymerization is Accompanied byFormation of Tyrosine Cross-Links

[0080] We initially tested whether peroxidase-catalyzed oxidativeconditions could promote Aβ polymerization by measuring the fluorescenceof human Aβ₁₋₄₀, human Aβ₁₋₄₂, and rat Aβ₁₋₄₀ (50 μM) incubated with orwithout H₂O₂ and peroxidase for 1 day. Fluorometric analysis of thesesamples indicated a marked increase in fluorescence in samplescontaining Aβ₁₋₄₀ and Aβ₁₋₄₂, as illustrated in FIG. 1A. These resultsare similar to those previously reported for synthetic human Aβ,achieved at a much higher peptide concentration, 1.25 mM (Galeazzi etal., 1999). In contrast to the behaviour of the human-sequence Aβpeptide, no increase in the fluorescence signal of rat Aβ₁₋₄₀ wasobserved after incubation with H₂O₂ and peroxidase, as also shown inFIG. 1A. This suggested that the fluorescent signal was specific fortyrosine-oxidation products of Aβ, since rat Aβ lacks tyrosine (Shiverset al., 1988).

[0081] To confirm that these reactions resulted in Aβ polymerization,Aβ₁₋₄₀ and Aβ₁₋₄₂ treated as described above were run on SDS-PAGE andanalyzed by Western blot. Both human synthetic Aβ₁₋₄₀ and Aβ₁₋₄₂incubated with H₂O₂ and peroxidase displayed marked increases inapparent SDS-resistant polymers compared to untreated Aβ, as shown inFIG. 1B. Neither polymerization nor increased fluorescence was observedwhen Aβ was incubated with either H₂O₂ or peroxidase alone.

EXAMPLE 2 Polymerization Occurs Under Physiological Conditions

[0082] To determine whether H₂O₂/peroxidase-induced polymerization ofsynthetic Aβ occurs under conditions which approached physiological, wealso incubated Aβ₁₋₄₂ at 10 nM with H₂O₂ at 1 μM and peroxidase (7.5μg/ml) in PBS at pH 7.4. We observed that SDS-resistance of the peptidewas again induced, as shown in FIG. 1C; however, oligomers of lowerapparent molecular weight than those generated by using higherconcentrations of substrates were generated, as illustrated in FIG. 1B.The migration on SDS-PAGE of the apparent Aβ polymers under theseconditions suggested the formation of dimers (8 Kd), trimers (13 kD,)and tetramers (17 kD).

[0083] As shown in FIG. 2A and FIG. 2B respectively, fluorescentanalysis of Aβ purified from AD-affected post-mortem brain tissuerevealed the characteristic spectrofluorometric pattern of tyrosinecross-linked species; this purified protein migrated as apparentoligomers on SDS-PAGE, as previously described (Roher et al., 1996).

EXAMPLE 3 Tyrosine Cross-Linking of Oligomers

[0084] To confirm that the apparently oligomeric human amyloid-derivedAβ was tyrosine cross-linked, a sample was hydrolyzed and then analyzedby MALDITOF-MS. This analysis, illustrated in FIG. 3A, indicated a peakcorresponding to 361 Da (m/z 361, representative of M+H), therebyconfirming the existence of DT or iso-DT in the sample. A smaller peakcorresponding to 540 Da was also detected, consistent with the presenceof TT or P. Other prominent peaks were detected at 247, 263, 307, 309and 538 Da; these may represent other modifications to Aβ amino acids,such as carbonylation (Atwood, 1999) and other amino acid cross-links.

[0085] More abundant fragments from the hydrolysis of human Aβ were alsodetected at 423 and 425 Da (ratio 3:2), suggestive of Cu binding to DTor iso-DT (Cu mass=63 & 65 Da, ≈2:1 natural isotope abundance).

EXAMPLE 4 Binding of Copper by Dityrosine

[0086] In order to test whether the peaks at 423 and 425 could be due toDT binding to Cu, we examined the interaction of Cu²⁺ with DT byspectroscopic analysis. Dityrosine (50 μM) was solubilized in phosphatebuffer (50 mM, pH 7.4) and the absorbance spectra (200-1000 nm) measuredon a SPECTRAmax Plus (Molecular Devices). A trough (280 nm) and peak(315 nm) were apparent. Dityrosine was then incubated with increasingconcentrations of CuNO₃ (0-200 μM) or NaCl (0-200 μM), and changes inabsorbance at both 280 nm and 315 nm were monitored.

[0087] We found that as DT was incubated with increasing concentrationsof Cu²⁺ its characteristic absorbance peak at 315 nm diminished, whereasa new absorbance peak developed at 280 nm. The spectroscopic changesreached a plateau at a stoichiometric ratios between 1:1-2:1 (Cu:DT),and then saturated at 3:1, suggesting that DT can bind up to 3equivalents of Cu. Dichloride binding would also produce a similar p+2mass unit increment (Cl mass=35 and 37 Da, ≈3:1 natural isotopeabundance), but coincubating DT with NaCl induced no spectroscopicabsorbance changes. These results are shown in FIG. 3C.

EXAMPLE 5 Dityrosination of Aβ Increases Its Copper-Binding Capacity

[0088] We predicted that a proportion of the Aβ found in the solublefraction of human brain would display enhanced copper binding propertiesdue to dityrosination. To test whether this was in fact the case, wepassed a portion of soluble extract of AD-affected brain over achelating Sepharose column charged with copper. 0.5 g of cerebral cortexgrey matter from frozen AD and control brains (AC) was homogenised in 3ml of ice cold phosphate buffered saline (PBS). Samples were centrifugedat 175 000 g for 1 hour and the supernatant retained for analysis of Aβcontent. 10 ml of supernatant was loaded onto a chelating Sepharosecolumn charged with 1 mg/ml copper sulphate. Unbound proteins werewashed through using a 0.05M Na acetate buffer with 0.5M NaCl at pH 8.The bound material eluted in a stepwise gradient of increasing acidity,using successive steps of pH 5.5, 3 and 1, followed by a wash with 50 mMEDTA to strip the column. Eluates were subjected. to exhaustive dialysisto remove free copper and salts using a size cutoff of 2 kDa,freeze-dried and subjected to SDS-PAGE, Western blot and LC-MS analyses.ESI mass spectra (+ve ion) were acquired on a Quatro II triplequadrupole (Micromass). Mass spectra were collected in continuum modeevery 8 seconds from 650 to 1650 m/z. Samples were introduced to the ionsource in 5 mM ammonium acetate buffer. Slot blot analysis showed no WO2immunoreactivity in the pH3 eluate, and a further elution was performedat pH 1. Strong immunoreactivity was detected at this pH, and thedialysed sample was blue in colour.

[0089] Western blot analysis revealed the presence of Aβ in the pH 1 andEDTA fractions; this suggested very high-affinity binding to copper,since pH 3 is usually sufficient to elute most copper-binding proteinfrom such a column. Material in these fractions was shown to be highlyenriched in oligomeric Aβ. These results are illustrated in FIG. 4.

[0090] Silver staining (FIG. 4A) demonstrated substantial metalaffinity-based purification (lane 1 vs. 2), and Western blot analysisdisplayed immunoreactive bands which appear to correspond to multiplesof monomeric Aβ (FIG. 4B). FIG. 5 shows LC (top) and MS (bottom) tracesfrom crude and IMAC-purified supernatant extracts from AD brain tissue.It is noticeable that the LC and MS spectra are substantially cleanerfor the IMAC purified sample. LC-MS analysis of the IMAC purified sampleproduced signals corresponding to Aβ species, including Aβ₁₋₄₀ bearing 2copper atoms, as confirmed by LC-MS analysis of synthetic peptide in thepresence or absence of copper. Highlighted peak clusters onrepresentative mass spectra indicate mass/charge ratios consistent withparent ions of masses 4515.1 (Aβ₁₋₄₂) and 4457.9 (Aβ₁₋₄₀ +2 Cu).

[0091] In order to confirm whether this strongly copper-binding Aβfraction contained DT, we employed the monoclonal antibody IC3 raisedagainst DT generated by a process using H₂O₂ and horseradish peroxidase(Kato et al. (1998); this was the gift of Dr. Yoji Kato of the HimejiInstitute of Technology, Himeji, Japan.). We found that the highermolecular weight oligomers of Aβ observed on Western blot co-localisedwith positive staining for DT.

[0092] The Aβ containing fractions also exhibited fluorescence emissionspectra characteristic of the presence of the dityrosine moiety. Thisemission was quenched by the addition of copper in a fashion predictedfor the enhanced copper binding due to this modification.

EXAMPLE 6 Further Characterisation of Dityrosinated Aβ

[0093] DT-enriched Aβ is isolated from the soluble fraction of humanbrain in sufficient quantity to carry out further characterisation.These studies include toxicity studies in tissue culture, amino acidsequencing, metal binding studies, and experiments to determine whetherDT-enriched Aβ has enhanced electrochemical activity, for exampleinduction of hydrogen peroxide formation and copper reduction.

EXAMPLE 7 Effect of Immunization against Dityrosine

[0094] We attempted to raise an immune response to DT in wild-type mice.In this experiment the DT was prepared by mixing tyrosine in boratebuffer with H₂O₂, and incubating this mixture with horseradishperoxidase, as described in the Experimental Procedures.

[0095] DT was conjugated to the carrier protein Keyhole LimpetHaemocyanin (KLH) using-glutaraldehyde and according to standardprotocols. An emulsion of each of DT-KLH, KLH alone or untreatedtyrosine was prepared in Freund's complete adjuvant, and two animalseach were inoculated intraperitoneally with an inoculum containing 100mg of either DT-KLH, or unreacted tyrosine or KLH alone. Pre-immuneserum was taken at this time. The first immune sera were collected 10days after immunization. Two booster immunizations were given atfortnightly intervals thereafter. Blood samples were taken at eachinoculation and at one week following the final boost.

[0096] An ELISA was adapted to assay the immune response to DT. We foundthat the immune responses to DT of the mice which were immunized witheither DT-KLH or unreacted tyrosine were never greater than theresponses of mice immunized with KLH alone. The DT monoclonal antibodyIC3 obtained from Dr. Kato was used as a positive control, and produceda modest positive reaction against DT in this assay.

[0097] In a second experiment, two rabbits were immunized with DT-KLH inthe manner described above. The ELISA results for sera produced by theseanimals demonstrated a moderate immune response against DT.

[0098] We also attempted to demonstrate the presence of endogenousantibodies to DT in individual sera from four human patients who werediagnosed with Alzheimer's disease by post mortem histopathology. Noimmunoreactivity against DT was observed in these sera by ELISA or byWestern blot.

[0099] In a further experimental iteration, we examined whether themouse or rabbit antisera raised against the DT-KLH described above,recognised DT moieties in the dimeric and higher order oligomers of Aβextracted from human brain. Surprisingly, none of the sera demonstratedactivity against DT moieties in human brain Aβ. The positive controlantibody IC3 was also negative in this assay.

EXAMPLE 8 Effect of the Method of Producing Dityrosine Moieties onImmunogenicity and Antibody Reactivity

[0100] We suspected that the unexpected lack of an immune response mightbe due to poor antigenicity of the dityrosine moieties.

[0101] To investigate this hypothesis, we prepared tyrosine cross-linkedsynthetic Aβ₁₋₄₀ and Aβ₁₋₄₂ by two different methods. The first methodinvolved incubation of the Aβ peptides in borate buffer with horseradishperoxidase and H₂O₂, as described in the Experimental Procedures above.

[0102] In the second method, a 2.5 μM solution of Aβ was prepared indouble deionised water containing 30 μM CuCl₂ and 200 μM H₂O₂, andincubated for one to five days at room temperature.

[0103] Samples of each variety of cross-linked Aβ were subjected toPAGE, and Western blotting was performed using the Aβ-specific antibodyWO2 or the positive control anti-DT antibody IC3. The results of theseblots are presented in FIG. 6.

[0104] The IC3 antibody detected DT in the cross-linked Aβ in both ELISAand Western blot assays. In addition, in Western blots the antibodyrecognised the presence of dityrosine in the DT-KLH produced in Example7. From these results it appears that Aβ₁₋₄₂ is more efficientlycross-linked by either the borate or copper methods than is Aβ₁₋₄₀. Inaddition, Aβ₁₋₄₀ loses immunoreactivity to WO2 when cross-linked withthe method involving copper. This may be due to greater susceptibilityof the peptide to free radical damage or the modification, masking orhindering of the antibody binding site after crosslinking.

[0105] Surprisingly, it is also evident from the differential stainingwith IC3 that the pattern of Aβ cross-linking through dityrosine dependson the different reactions used to produce the crosslinking. The IC3monoclonal antibody did not detect DT produced by the boric acid method,but did detect DT produced by the copper method.

[0106] Also surprisingly, the IC3 antibody detected DT cross-linking inAβ₁₋₄₀ in preference to Aβ₁₋₄₂. This pattern is the inverse of thatobserved with the anti Aβ antibody WO2.

[0107] These results demonstrate that the method of inducing DTcross-linking and the structure of the polypeptide being cross-linkedare crucial variables in recognition of DT by an antibody. In this case,the addition of two amino acid residues to dityrosine-linked Aβ₁₋₄₀resulted in a dramatic decrease in the ability of an anti-dityrosineantibody to bind. This result may be extrapolated to the in vivosituation, suggesting that the selection of antigen is critical toeliciting a physiologically-relevant immune response.

EXAMPLE 9 Effect of the Form of Tyrosine Cross-Link on AntibodyRecognition

[0108] It was anticipated that a DT inoculum must be conjugated to alarge carrier protein to provoke an immune response. Furthermore, thequality of the immune response generated would also be in part dependentupon the selection of an appropriate carrier. To examine this weselected two alternative carriers for various DT species, Bovine SerumAlbumin (BSA) and Keyhole Limpet Haemocyanin (KLH).

[0109] In addition, to investigate the role of different forms ofdityrosine in immuno-recognition, we prepared a crude mixture whichcontained variety of forms of DT, including numerous oligomers andbranched forms of DT. The tyrosine cross-links in this crude mixturewere created using the borate/H₂O₂/peroxidase method described above.The resulting DT mixture contained molecules with linkages at a varietyof positions on the ring and backbone of the tyrosine molecule. Examplesof the structures produced are illustrated in FIG. 7.

[0110] The crude mixture was then separated by reverse phase HPLC intofractions which contained predominantly mono-dityrosine, dityrosine,trityrosine and polytyrosine.

[0111] Two important characteristics of the oligomeric structures arethat they can present multiple copies of desired antigen to improveimmunogenicity and enhance the immune response, and that they can allowthe presentation of alternative forms of chemical bonds between thetyrosine residues.

[0112] To investigate the nature of the tyrosine cross-links whichcomprise the oxidative modifications to Aβ in vivo in AD, we alsoprepared tyrosine cross-linked Aβ fragments. Using the same technique,we prepared molecules consisting of two or more Aβ₉₋₁₆ peptide chainscross-linked by dityrosine (structures not shown). The resultantcross-links most probably represent a racemic mixture of a variety offorms of tyrosine cross-links.

[0113] A number of the novel structures described above werecharacterised in Western blots using the anti-DT monoclonal IC3 or theimmune serum from a rabbit which was immunized with DT-KLH (described inExample 7). These results are presented in FIG. 8.

[0114] The results demonstrated that the dimer but not the trimer ofAβ₉₋₁₆ linked to BSA was immunoreactive to both the rabbit immune serumand the monoclonal antibody IC3.

[0115] The presence of KLH was recognised by the rabbit immune serum inthe blots irrespective of whether it was conjugated to an additionaltyrosine cross-link antigen. Polytyrosine-BSA and polytyrosine-KLH wererecognised by IC3, but the rabbit immune serum could not distinguishbetween KLH alone and polytyrosine-KLH.

[0116] It is clear from these results that the rabbit immunizationelicited an antibody which was reactive with some forms of dityrosinebut not others, as predicted from the data presented in FIG. 6.

EXAMPLE 10 Effect of Immunization with Dityrosine on Aβ Deposits inTransgenic Animals

[0117] Transgenic mouse models are available for a number ofneurological disorders, including Alzheimer's disease (Games et al.,1995; Hsiao et al., 1996); Parkinson's disease (Masliah et al., 2000);familial amyotrophic lateral sclerosis (ALS) (Gurney et al., 1994);Huntington's disease (Reddy et al., 1998); and Creutzfeld-Jakob disease(CJD) (Telling et al., 1994).

[0118] We have found that one of the transgenic models for Alzheimer'sdisease, the APP2576 tg mouse (Hsiao et al., 1996) also has a highincidence of cataract. These animal models are suitable for testing themethods of the invention.

[0119] Transgenic mice of the Strain APP2576 (Hsiao et al 1996) areused. Eight to nine month old female mice are selected and divided intogroups for treatment.

[0120] Tyrosine cross-linked antigens are prepared using a variety oftechniques to generate different forms of tyrosine cross-links. Antigensused include: Antigen Carrier protein Aβ₉₋₁₆ dimer BSA Aβ₉₋₁₆ trimer BSA(crude) ATEE BSA poly-tyrosine BSA Aβ trimer KLH (crude) ATEE KLHpoly-tyrosine KLH

[0121] Each immunisation comprises 25 μg of antigen in Freund's completeadjuvant, in a total volume of 0.5 ml, given subcutaneously.

[0122] Control animals received carrier protein without the tyrosinecross-linked antigen.

[0123] Samples of serum are taken at 14 day intervals, with boosterimmunizations given at 28 days. Serum samples are assayed for thepresence of anti-DT antibody, using the ELISA method of Kato et al forexample. It is expected that high antibody titres are obtained by aboutfive weeks following the final booster injection. The levels of Aβ inthe blood are also determined.

[0124] Once high titre antibody is present, mice are sacrificed atintervals, and their brains examined to determine whether theimmunization decreases brain amyloid formation, and to identify the mosteffective immunization protocol. The levels of soluble and insoluble Aβin the brain and serum is determined using calibrated Western blots. TheAβ plaque burden in the brain is examined immunohistochemically.

[0125] Other mice in each group are tested over a period of up to eightmonths for cognitive performance using a Morris water maze according tostandard methods. The general health and well being of the animals isalso measured every day by a blinded operator using a five point integerscale that subjectively rates a combination of features including motoractivity, alertness and general health signs.

EXAMPLE 11 Effect of Treatment with Antibodies against Dityrosine

[0126] Normal mice are hyperimmunized by standard procedures well knownin the art with one or more of the immunogens described in Example 7.The mice are bled at intervals and their sera assayed for anti-DT asdescribed above. Upon detection of high titre antibody, sera areharvested and the antibody component isolated and/or enriched usingmethods commonly available in the art.

[0127] These antibodies are injected intravenously or directly into theCSF of APP2576 transgenic mice, either in a single dose or repeateddosages over a course of days or weeks.

[0128] The transgenic mice are sacrificed at intervals followingtreatment with anti-dityrosine antibodies, and their brains examined todetermine whether antibody treatment decreases brain amyloid formation.

EXAMPLE 12 Diagnosis of Conditions Associated with TyrosineCross-Linking

[0129] Samples of sera and cerebrospinal fluid (CSF) from patientsconfirmed to be suffering from AD and from age-matched controls areassayed for the presence of tyrosine cross-linked compounds usingfluorescence analysis as described above. In one set of samples,tyrosine cross-linked compounds in the sample are first enriched bypassing the sample over a solid support coupled to nitrilotriaceticacid, as described in U.S. Pat. No. 5,972,674.

[0130] Similar assays are performed using samples from patents sufferingfrom ALS, Parkinson's disease, and CJD.

[0131] It is possible that patients may also have circulating antibodiesdirected against tyrosine cross-linked compounds, and so in analternative assay such antibodies are directed in either sera or CSFusing an ELISA assay, employing monoclonal antibodies directed againstDT (Kato et al., 1998).

EXAMPLE 13 Identification of the Forms of Dityrosine Present inOxidatively-Modified Aβ

[0132] In order to identify the predominant form or forms of DT presentin oxidatively modified Aβ, enzymatic digestion fragments ofcopper-catalysed Aβ oligomers are generated, and the fragments analysedby mass spectrometry. This technique has recently been applied to theanalysis of copper-catalysed oxidative modifications to the prionprotein (Requena, J. R., et al. 2001 PNAS 98: 7170-7175)

[0133] This enables the identification of the antigen most likely to beeffective in eliciting monoclonal antibodies suitable for use in passiveimmunization, as described in Example 11. Methods for generating highlyspecific monoclonal antibodies against any specific antigen are wellknown in the art. Once the antigen has been selected, a systematicanalysis of the most effective means of antigen presentation is carriedout using known methods.

[0134] Discussion

[0135] The neuronal damage in AD is associated with soluble Aβ ratherthan insoluble Aβ which is immobilised in neuritic plaques (McLean etal., 1999). We have now shown for the first time that the neurotoxic Aβoligomers extracted from AD-affected brains contain tyrosinecross-links), which may be DT, iso-DT, TT and/or P. These modificationswere emulated in vitro by incubating Aβ with peroxidase and H₂O₂, or byoxidation of Aβ in the presence of copper ions. These modificationscould interfere with the metabolism of Aβ, may contribute to theneurotoxicity seen in AD, and is indicative of the neurochemicalderangement in the disease.

[0136] The formation of the carbon-carbon bridge between DT, T and P isthought to be irreversible; DT cross-links are very resistant tohydrolytic cleavage by 6N HCl at 110° C. for 24 h, and to proteasedigestion (Smail et al., 1995). Pathologically, the catabolic resistanceof DT modifications of proteins could explain the contribution oftyrosine polymers to lipofuscin formation (Kato et al., 1998), and tothe cross-linking of α-crystallin in fluorescent cataract formation(Kikugawa et al., 1991). Clearly, tyrosine cross-linkage of Aβ would beexpected to inhibit its catabolism, and so may be an important step inthe evolution of amyloid plaque deposits in AD.

[0137] The formation of tyrosine cross-links necessitates that moleculescontaining tyrosyl radicals come into contact. Our results suggest thatthe tyrosine residue of Aβ must be accessible to peroxidase(s), and thattyrosyl residues between Aβ subunits of amyloid must, at some stage, bein apposition.

[0138] Since H₂O₂ is required for DT formation, the detection of DTmodifications in AD-derived brain Aβ implies that H₂O₂ is elevated inthe brain in AD. Without wishing to be bound by any proposed mechanism,we believe that phagocytic activation of the microglial cells in thebrain parenchyma, which is closely associated with amyloid formation inAD (Sheng et al., 1997), could contribute peroxidase activity and H₂O₂to cause tyrosine cross-linkage of Aβ. Activated rat microglia have beenobserved to have increased peroxidase levels (Lindenau et al., 1998),and in vitro experiments have demonstrated the capacity of Aβ to primeand/or trigger the respiratory burst of cultured rat microglia and humanphagocytes (Van Muiswinkel et al., 1996). Activated phagocytes releasemyeloperoxidase (Pember et al., 1983), and generate reactive oxygenspecies during the respiratory burst. This response is designed to killinvading pathogens or tumor cells; however, this environment has alsobeen shown to promote the oxidation of surrounding proteins and lipids(Byun et al., 1999). A similar microenvironment may be generated in thevicinity of activated microglia. In vitro, myeloperoxidase-H₂O₂ systemspromote the synthesis of tyrosine cross-linked species such as DT, TT, Pand isoDT (Jacob et al., 1996).

[0139] Thus the activation of microglia in response to Aβ accumulationmay promote tyrosine cross-linkage of the Aβ, inhibiting its clearanceand leading to a vicious cycle. Contributing to this possible viciouscycle, a proximate source of H₂O₂ for DT formation may be generated byAβ itself, since Aβ forms H₂O₂ by reacting with O₂ through the reductionof substoichiometric amounts of Cu²⁺ or Fe²⁺ (Huang, Atwood, et al.,1999; Huang, Cuajungco, et al., 1999). Therefore, it is highlysignificant that Aβ was purified intact, together with bound copper,from human amyloid (FIG. 3A). Synthetic Aβ₁₋₄₂ binds Cu²⁺ with attomolaraffinity, and since-copper is enriched in AD amyloid (Lovell et al.,1998), we had suspected that Aβ might bind copper in vivo. The findingthat amyloid-derived Aβ contains copper is also relevant to ADpathophysiology, because Cu²⁺ precipitates Aβ (Atwood et al., 1998), andthe toxicity of the peptide is potentiated by Cu²⁺ (Huang, et al.,1999).

[0140] Intriguingly, Cu²⁺ remained bound to DT after acid hydrolysis ofthe human amyloid-derived Aβ, as well as under the acidic conditions ofthe mass spectrometry (FIG. 3A). This unusual affinity for Cu²⁺ could bethe result of an adventitious high-affinity Cu²⁺ binding site on Aβbeing formed by the DT modification. As a consequence of thisexaggerated affinity for Cu²⁺, the neurotoxicity of DT-modified Aβ orits electrochemical activity may be increased compared to non-modifiedAβ. Adventitious Cu²⁺ binding caused by the DT modification could alsoexaggerate the precipitation of Aβ into amyloid, which would explain whytreatment with chelators at pH 7.4 promoted the release of dimeric Aβ toa greater extent than that of monomeric Aβ (assayed by Western blot)from post-mortem AD brain tissue (Cherny et al., 1999). The combinationof increased proteolytic resistance and adventitious metal binding maybe particularly pernicious consequences of the tyrosine cross-linking ofAβ which contribute to the pathology of AD.

[0141] PDAPP transgenic mice overproduce the human form of Aβ₁₋₄₂ andshow extensive cerebral amyloid plaque deposition with aging, as well asbehavioural and cognitive deficits (Games et al., 1995; WO96/40896).Immunisation of mature PDAPP mice with synthetic Aβ₁₋₄₂ results in astriking diminution in the number and intensity of amyloid plaques,while PDAPP mice immunised with this antigen fail to develop amyloidplaques (Schenk et al., 1999 and WO99/27944). It appeared that asuccessful immune response to Aβ₁₋₄₂ had been induced, with evidence ofscavenging microglial cells in the immediate vicinity of the remnantamyloid plaques, and the presence in blood of antibodies directedagainst Aβ₁₋₄₂ The authors suggested that immunization with Aβ could beused for prevention or treatment of AD. However, it is widely thoughtthat it is unlikely that an immunotherapy for AD is feasible, because ahuman recipient would be unable to mount a significant immune responseto a self protein because of immunological tolerance. The resultsobtained by Schenk et al. suggest that the brain may have the capacityto resorb and clear otherwise intractable amyloid deposits, given theappropriate stimulus. However, it is undesirable to use immunisationwith Aβ itself, because of the potential for induction of harmfulautoimmune responses, and/or the induction of an inadequate, nonplaque-clearing response. By immunising with non-native dityrosine ordityrosine-containing compounds according to the present invention, thisproblem can be avoided.

[0142] It will be apparent to the person skilled in the art that whilethe invention has been described in some detail for the purposes ofclarity and understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this specification.

[0143] References cited herein are listed on the following pages, andare incorporated herein by this reference.

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1. A method of prophylaxis, treatment or alleviation of a condition,comprising the step of immunizing a subject in need thereof with animmunizing-effective dose of one or more tyrosine cross-linkedcompounds, wherein the condition is characterised by pathologicalaggregation and accumulation of a specific protein associated withoxidative damage and formation of tyrosine cross-links.
 2. A methodaccording to claim 1, in which the pathologically aggregated form of thespecific protein comprises a tyrosine cross-linked moiety.
 3. A methodaccording to claim 2, in which the tyrosine cross-linked compound is apeptide which is an immunogenic portion of the pathologically aggregatedform of the specific protein, the peptide comprising a cross-linkedtyrosine moiety linked to residues upstream and downstream of thecross-linked tyrosine.
 4. A method according to claim 1, in which thetyrosine cross-links in the compound are obtainable by oxidation in thepresence of copper ions.
 5. A method according to claim 1, in which thetyrosine cross-linked compound is a dityrosine cross-linked compound. 6.A method according to claim 1, in which the tyrosine cross-linkedcompound is complexed with copper ions.
 7. A method according to claim6, in which the ratio of copper ions to dityrosine is less than or equalto 3:1, provided that each dose administered contains no more than 1 μMcopper.
 8. A method according to claim 1 in which the compound iscoupled to a carrier protein which is itself immunogenic.
 9. A methodaccording to claim 8 in which the carrier protein is selected from thegroup consisting of tetanus toxoid, keyhole limpet haemocyanin, andalbumin.
 10. A method according to claim 1 in which the compound isadministered together with an adjuvant.
 11. A method according to claim3, in which the immunogenic portion of the specific protein is theminimal portion which is immunogenic.
 12. A method according to claim 1,in which the condition is Alzheimer's disease, the specific protein isAβ, and the tyrosine cross-linked compound is a tyrosine cross-linkedpeptide derived from the sequence surrounding tyrosine 10 in the aminoacid sequence of human Aβ₁₋₄₀ or Aβ₁₋₄₂.
 13. A method according to claim12, in which the tyrosine cross-linked peptide comprises dityrosine. 14.A method according to claim 12 in which the tyrosine cross-linkedpeptide is complexed with copper ions.
 15. A method according to claim1, comprising the additional steps of: identifying the predominant formsof the tyrosine cross-links in the pathologically aggregated specificprotein; and synthesising one or more tyrosine cross-linked compoundscomprising one or more of the predominant forms of tyrosine cross-links.16. A method of prophylaxis, treatment or alleviation of a condition, inwhich the condition is characterised by pathological aggregation andaccumulation of a specific protein associated with oxidative damage andwhere the pathologically aggregated form of the specific proteincomprises a tyrosine cross-link, the method comprising the step ofadministering an effective amount of an antibody or an antibodyfragment, said antibody or antibody fragment is raised against atyrosine cross-linked compound, said compound being an immunogenicportion of the pathologically aggregated form of the specific proteinand comprising a tyrosine cross-link, and which antibody or antibodyfragment is capable of specifically binding the pathologicallyaggregated form of the specific protein, to a subject in need of suchtreatment.
 17. A method according to claim 16, in which the antibody ispolyclonal.
 18. A method according to claim 16, in which the antibody isof human origin.
 19. A method according to claim 16, in which theantibody is monoclonal.
 20. A method according to claim 19, in which theantibody is humanized.
 21. A method according to claim 16, in which theantibody or antibody fragment reacts specifically with thepathologically aggregated form of the specific protein, and does notreact significantly with the unaggregated form of the specific protein.22. A method according to claim 16, in which the tyrosine cross-links inthe compound are obtainable by oxidation in the presence of copper ions.23. A prophylactic or therapeutic composition for use in a methodaccording to claim 1, comprising a tyrosine cross-linked compound,together with a pharmaceutically acceptable carrier.
 24. A prophylacticor therapeutic composition according to claim 23, in which the tyrosinecross-linked compound is complexed with copper ions.
 25. A prophylacticor therapeutic composition according to claim 23, further comprising anadjuvant.
 26. A prophylactic or therapeutic composition for use in amethod according to claim 16, the composition comprising an antibody oran antibody fragment raised against a tyrosine cross-linked compound,said compound being an immunogenic portion of the pathologicallyaggregated form of the specific protein and comprising a tyrosinecross-link, and which antibody or antibody fragment is capable ofspecifically binding the pathologically aggregated from of the specificprotein, together with a pharmaceutically acceptable carrier.
 27. Amethod of diagnosis of a condition selected from the group consisting ofAlzheimer's disease, amyotropic lateral sclerosis, motoneuron disease,Creutzfeldt-Jacob disease and Huntington's disease, comprising the stepof assaying a sample of a biological fluid from a subject suspected ofsuffering from the condition for the presence of a molecule comprisingtyrosine cross-links.
 28. A method of diagnosis of a condition selectedfrom the group consisting of Alzheimer's disease, amyotropic lateralsclerosis, motoneuron disease, Creutzfeldt-Jacob disease andHuntington's disease, comprising the step of assaying a biological fluidfrom a subject suspected of suffering from the condition for thepresence of antibody directed against a molecule comprising tyrosinecross-links.
 29. A method according to claim 27 or claim 28, in whichthe molecule comprises dityrosine.
 30. A method according to claim 27 orclaim 28, in which the biological fluid is selected from the groupconsisting of blood, plasma, serum, cerebrospinal fluid, urine, andsaliva.
 31. A method according to claim 1, claim 16, claim 17 or claim28, in which the condition is Alzheimer's disease.